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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-08-19 to 2009-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002-04-24
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
cited as Commission Regulation (EC) No 440/2008, 2008-05-30
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,3,6-Trimethylphenol
- Analytical purity: 99.8 area-%
- Lot/batch No.: 56377456P0
- Homogeneity: The test item was homogeneous by visual inspection
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by BASF SE

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
female CBA/J mice
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.3 - 21.9 g
- Housing: single housing in Makrolon cages, type II
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 days before the first test-item application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2009-08-19 (arrival of the animals at the testing facility) To: 2009-09-07 (sacrifice)

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Remarks:
(MEK)
Concentration:
10, 30, 60% (w/w)
No. of animals per dose:
5 females per group
Details on study design:
RANGE FINDING TESTS:
- The selection of concentrations took into account available information on the chemical/physical properties, the composition and on acute toxicity and primary irritation/corrosion potential of the test item. In addition the results of a pretest with a 60% test-item preparation were considered, which did not show increased ear weights, but increased lymph node weights as indication of ear irritation. Due to the chemical/physical properties of the test substance (melt), higher concentrations could not be applied technically.
- The following dose levels were selected: 10%, 30% and 60% in methyl ethyl ketone (MEK). A vehicle control group was included.
MEK was used as the vehicle because good solubility of the preparation was achieved.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, ³H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
The increase SI of cell count by a factor of ≥ 1.5 and/or of ³H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

In addition the evaluation uses the following considerations:
If biologically relevant increases in ear weights are running in parallel to the increase in cell count, ³H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Depending on the magnitude of lymph node response the evaluation of the sensitizing potential may be modified or additional studies might be necessary.
If a test substance does not elicit a biological relevant increase in cell count, ³H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
For better handling the test item was heated at ca. 80°C before test-item preparation. The test-item preparations were produced on a weight per weight basis shortly before the application. After stirring with a magnetic stirrer the test item was soluble in the vehicle.
The study used 4 groups of 5 female CBA/J mice each (3 test groups and 1 control group). The mice were treated with 10%, 30% and 60% (w/w) preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone. The 60% preparation was the maximum technically applicable concentration.
Each test animal was applied with 25 µL per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 µCi of ³H-thymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and ³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm (ca. 50 mm²) was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

Calculations:
The stimulation indices of cell count, ³H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

Further examinations:
Body weight determination:
Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.

Signs and symptoms:
No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted.

Mortality:
Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.

Positive control:
A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Separate studies using the positive control substance alpha-hexylcinnamaldehyde are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

Results and discussion

Positive control results:
Positive control study: Alpha-Hexylcinnamaldehyde, techn. 85%; Group mean values: Cell Counts/ Lymph Node Pair: 14,832,000; ³H-thymidine
incorporation [DPM/Lymph Node Pair]: 2,270.5; Lymph Node Weight [mg//Lymph Node Pair]; Ear Weight [mg/animal].
see attached file

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI values calculated for ³H-TdR incorporation: vehicle control: 1.00 10% in MEK: 1.11 30% in MEK: 2.34 60% in MEK: 3.62 For other stimulation indices, see freetext in "Remarks on results including tables and figures", table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: ³H-thymidine incorporation [DPM/Lymph Node Pair] vehicle control: 702.6 10% in MEK: 777.9 30% in MEK: 1647.3 60% in MEK: 2542.2 see also attached file.

Any other information on results incl. tables

The stimulation indices (fold of change as compared to the vehicle control) for cell count, ³H-thymidine incorporation, lymph node weight and ear weight are summarized for each test group in table 2.

 

Table 2: Simulation indices

 

Test

group

Treatment

Cell Count

Stimulation

Index

³H-thymidine

incorporation

Stimulation Index

Lymph Node

Weight

Stimulation Index

Ear Weight

Stimulation

Index

1

vehicle MEK

1.00

1.00

1.00

1.00

2

10% in MEK

1.04

1.11

1.10

1.00

3

30% in MEK

1.26

2.34

1.27

1.04

4

60% in MEK

1.83

3.62

1.62

1.15

 

No signs of systemic toxicity were noticed.

When applied as 60% preparation in MEK, the test item induced a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

There was an increase in lymph node weights, as well.

Concomitantly, the increase of 3H-thymidine incorporation into the cells was biologically relevant (increase above the cut off stimulation index of 3) at this concentration.

The 60% test-item preparation caused some increase in ear weights as indication of ear skin irritation, the magnitude of which, however, does not fully explain the measured lymph node reactions.

The increases in cell count,3H-thymidine incorporation, lymph node weight and ear weight were concentration dependent.

 

Conclusion:

The authors concluded that 2,3,6-Trimethylphenol shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >30%<60%. The estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for  3H-thymidine incorporation (EC 3) was calculated by linear regression from the results of the 30% and 60% concentrations to be 42.6% and 45.4%, respectively.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Based on the results of this test, it is concluded that 2,3,6-trimethylphenol shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.